DNA/RNA Purity (260/280 and 260/230) Calculator
Compute DNA or RNA concentration from A260 plus the 260/280 and 260/230 purity ratios — with A320 background correction, path length, dilution, yield, molarity and copies per µL, a dilution recipe and a ready-or-clean-up check for PCR, qPCR, sequencing, cloning and transfection.
How it works
Concentration (ng/µL) = (A260 − A320) ÷ path (cm) × factor × dilution, factor = 50 (dsDNA), 40 (RNA), 33 (ssDNA/oligo) µg/mL per A260 · Purity = (A260 − A320) ÷ (A280 − A320) and (A260 − A320) ÷ (A230 − A320) · Yield = concentration × volume · MW: dsDNA = bp × 617.96 + 36.04, ssDNA = nt × 308.97 + 18.02, RNA = nt × 320.5 + 159 · nM = ng/µL ÷ MW × 10⁶ · copies/µL = nM × 6.022 × 10⁸
Nucleotides absorb at 260 nm, proteins at 280 nm, and salts, phenol, guanidine and carbohydrates near 230 nm. One absorbance unit at 260 nm in a 1 cm path is about 50 µg/mL of double-stranded DNA, 40 µg/mL of RNA and 33 µg/mL of single-stranded DNA; oligos vary with sequence, so use the supplier’s µg per OD as a custom factor. A260/A280 of about 1.8 for DNA (2.0 for RNA) and A260/A230 of 2.0–2.2 indicate a clean prep. An A320 reading, where nucleic acids do not absorb, measures turbidity or bubbles and is subtracted from every wavelength before the maths. Readings are most trustworthy between about 0.1 and 1.5; below that the ratios become noisy. A low ratio rarely changes the concentration much, but it can inhibit enzymes downstream: PCR tolerates a lot, while sequencing library prep and transfection do not — the readiness table applies common rule-of-thumb targets. Enter the fragment length to get the molar concentration and copies per µL, and a target concentration to get a dilution recipe.
Worked example
A260 0.42, A280 0.23 and A230 0.20 for undiluted dsDNA: 0.42 × 50 = 21.0 ng/µL; 260/280 = 1.83 (pure) and 260/230 = 2.10 (clean). In 50 µL that is 1.05 µg, and 1 µg needs 47.62 µL. For a 3,000 bp plasmid the molecular weight is 3,000 × 617.96 + 36.04 = 1,853,916 g/mol, so 21 ng/µL is 11.33 nM, or 6.82 × 10⁹ copies per µL. To make 50 µL at 10 ng/µL, mix 23.81 µL of sample with 26.19 µL of water.
Sources: Promega — How do I determine the concentration, yield and purity of a DNA sample? · QIAGEN — dsDNA mass to moles converter (617.96 g/mol per bp + 36.04)
Frequently asked questions
My 260/280 is 1.6 — is the DNA unusable?
Usually fine for PCR; clean up with a column or ethanol precipitation before sequencing, NGS or transfection. The readiness table shows which uses your ratios meet.
Why does A230 matter more for RNA kits?
Guanidine thiocyanate from lysis buffers absorbs strongly at 230 nm and inhibits reverse transcription, so a low 260/230 is a common reason RT-qPCR fails. An extra wash with 80% ethanol on the column usually helps.
What is the A320 correction?
Nucleic acids and proteins do not absorb at 320 nm, so any reading there comes from turbidity, bubbles or a dirty cuvette. Subtracting A320 from A260, A280 and A230 removes that offset; many instruments do it automatically.
How do I convert ng/µL to nM or copies?
Divide by the molecular weight: for dsDNA about 618 g/mol per base pair. Enter the length under More options and the calculator gives nM and copies per µL — useful for ligation ratios, library pooling and qPCR standards.
What path length should I enter for a NanoDrop?
1 cm. The NanoDrop measures over 1 mm or less but reports absorbance scaled to a 10 mm path. For a plate reader without path correction, enter the real liquid height in the well (often 0.3–0.6 cm).
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